title: Targeting the AKT/GSK3β/cyclin D1/Cdk4 survival signaling pathway for eradication of tumor radioresistance acquired by fractionated radiotherapy.
abstract: OBJECTIVE
  Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3β-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3β/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells.
  
  
  METHODS
  Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice.
  
  
  RESULTS
  Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression.
  
  
  CONCLUSIONS
  Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3β/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor eradication in combination with chemotherapy.
authors.affiliation: Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Shimura
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kakuda
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ochiai
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Kuwahara
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Takai
authors.forename: Manabu
authors.initials: M
authors.lastname: Fukumoto
mesh_terms: D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002747:Chlorpropamide / Q000031:analogs & derivatives / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D019938:Cyclin D1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4 / Q000037:antagonists & inhibitors*; D019583:Dose Fractionation, Radiation; D015536:Down-Regulation; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D011836:Radiation Tolerance / Q000187:drug effects* / Q000502:physiology / Q000528:radiation effects; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D023041:Xenograft Model Antitumor Assays / Q000379:methods; D000096926:Benzenesulfonamides
chemical_list: D002747:Chlorpropamide; D002945:Cisplatin; D019938:Cyclin D1; D051358:Cyclin-Dependent Kinase 4; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D009363:Neoplasm Proteins; D051057:Proto-Oncogene Proteins c-akt; D011838:Radiation-Sensitizing Agents; C111224:API 2; D000096926:Benzenesulfonamides; C000605506:GSK3B protein, human; C000605507:Gsk3b protein, mouse